The present invention relates generally to the identification and isolation of novel DNA and to the recombinant production of novel polypeptides having homology to connective tissue growth factor, designated herein as Wnt-1-Induced Secreted Proteins (WISPs).
Malignant tumors (cancers) are the second leading cause of death in the United States, after heart disease. Boring et al., CA Cancer J. Clin., 43: 7 (1993).
Cancer is characterized by the increase in the number of abnormal, or neoplastic, cells derived from a normal tissue which proliferate to form a tumor mass, the invasion of adjacent tissues by these neoplastic tumor cells, and the generation of malignant cells which eventually spread via the blood or lymphatic system to regional lymph nodes and to distant sites (metastasis). In a cancerous state a cell proliferates under conditions in which normal cells would not grow. Cancer manifests itself in a wide variety of forms, characterized by different degrees of invasiveness and aggressiveness.
Alteration of gene expression is intimately related to the uncontrolled cell growth and de-differentiation which are a common feature of all cancers. The genomes of certain well studied tumors have been found to show decreased expression of recessive genes, usually referred to as tumor suppression genes, which would normally function to prevent malignant cell growth, and/or overexpression of certain dominant genes, such as oncogenes, that act to promote malignant growth. Each of these genetic changes appears to be responsible for importing some of the traits that, in aggregate, represent the full neoplastic phenotype. Hunter, Cell, 64: 1129 (1991); Bishop, Cell, 64: 235-248 (1991).
A well-known mechanism of gene (e.g., oncogene) overexpression in cancer cells is gene amplification. This is a process where in the chromosome of the ancestral cell multiple copies of a particular gene are produced. The process involves unscheduled replication of the region of chromosome comprising the gene, followed by recombination of the replicated segments back into the chromosome. Alitalo et al., Adv. Cancer Res., 47: 235-281 (1986). It is believed that the overexpression of the gene parallels gene amplification, i.e., is proportionate to the number of copies made.
Proto-oncogenes that encode growth factors and growth factor receptors have been identified to play important roles in the pathogenesis of various human malignancies, including breast cancer. For example, it has been found that the human ErbB2 gene (erbB2, also known as her2, or c-erbB-2), which encodes a 185-kd transmembrane glycoprotein receptor (p185HER2; HER2) related to the epidermal growth factor receptor (EGFR), is overexpressed in about 25% to 30% of human breast cancer. Slamon et al., Science, 235:177-182 (1987); Slamon et al., Science, 244:707-712 (1989). It has been reported that gene amplification of a protooncogen is an event typically involved in the more malignant forms of cancer, and could act as a predictor of clinical outcome. Schwab et al., Genes Chromosomes Cancer, 1: 181-193 (1990); Alitalo et al., supra. Thus, erbB2 overexpression is commonly regarded as a predictor of a poor prognosis, especially in patients with primary disease that involves axillary lymph nodes (Slamon et al., (1987) and (1989), supra; Ravdin and Chamness, Gene, 159:19-27 (1995); and Hynes and Stern, Biochim Biophys Acta, 1198:165-184 (1994)), and has been linked to sensitivity and/or resistance to hormone therapy and chemotherapeutic regimens, including CMF (cyclophosphamide, methotrexate, and fluoruracil) and anthracyclines. Baselga et al., Oncology, 11(3 Suppl 1):43-48 (1997). However, despite the association of erbB2 overexpression with poor prognosis, the odds of HER2-positive patients responding clinically to treatment with taxanes were greater than three times those of HER2-negative patients. Baselga et al., supra. A recombinant humanized anti-ErbB2 (anti-HER2) monoclonal antibody (a humanized version of the murine anti-ErbB2 antibody 4D5, referred to as rhuMAb HER2 or HERCEPTIN(copyright)) has been clinically active in patients with ErbB2-overexpressing metastatic breast cancers that had received extensive prior anticancer therapy. Baselga et al., J. Clin. Oncol., 14:737-744 (1996).
Cytokines have been implicated in the pathogenesis of a number of brain diseases in which neurological dysfunction has been attributed to a change in amino acid neurotransmitter metabolism. In particular, members of the transforming growth factor-xcex2 (TGF-xcex2) family have been implicated. TGF peptides are small polypeptides that were first identified by their ability to induce proliferation and transformation in noncancerous cells in culture. Although initially defined as a growth factor, TGF-xcex2 also inhibits proliferation of epithelial, endothelial, lymphoid, and hematopoietic cells. This cytokine is thought to play an important role in regulating the duration of the inflammatory response, allowing the healing process to proceed. It is also a potent immunomodulator, which has many pleiotrophic effects, including regulating many other cytokines.
The TGF-xcex2 superfamily includes bone morphogenetic proteins (BMP-2, BMP-4, BMP-5, BMP-6, BMP-7), activins A and B, decapentaplegic (dpp), 60A, OP-2, dorsalin, growth differentiation factors (GDFs), nodal, MIS, Inhibin-xcex1, TGF-xcex21, TGF-xcex22, TGF-xcex23, TGF-xcex25, and glial-derived neurotrophic factor (GDNF). Atrisano, et al., J. Biochemica et Biophysica Acta, 1222:71-80 (1994). Of particular interest are the growth differentiation factors, for as their name implies, these factors are implicated in the differentiation of cells.
Connective tissue growth factor (CTGF) is a growth factor induced in fibroblasts by many factors, including TGF-xcex2, and is essential for the ability of TGF-xcex2 to induce anchorage-independent growth (AIG), a property of transformed cells. CTGF was discovered in an attempt to identify the type of platelet-derived growth factor (PDGF) dimers present in the growth media of cultured endothelial cells, and is related immunologically and biologically to PDGF. See U.S. Pat. No. 5,408,040. CTGF also is mitogenic and chemotactic for cells, and hence growth factors in this family are believed to play a role in the normal development, growth, and repair of human tissue.
Seven proteins related to CTGF, including the chicken ortholog for Cyr61, CEF10, human, mouse, and Xenopus laevis CTGF, and human, chicken, and Xenopus laevis Nov have been isolated, cloned, sequenced, and characterized as belonging to the CTGF gene family. Oemar and Luescher, Arterioscler. Thromb. Vasc. Biol., 17: 1483-1489 (1997). The gene encoding Cyr61 has been found to promote angiogenesis, tumor growth, and vascularization. Babic et al., Proc. Natl. Acad. Sci. USA, 95: 6355-6360 (1998). The nov gene is expressed in the kidney essentially at the embryonic stage, and alterations of nov expression, relative to the normal kidney, have been detected in both avian nephroblastomas and human Wilms"" tumors. Martinerie et al., Oncoaene, 9: 2729-2732 (1994). Wt1 downregulates human nov expression, which downregulation might represent a key element in normal and tumoral nephrogenesis. Martinerie et al., Oncogene, 12: 1479-1492 (1996). It has recently been proposed that the CTGF, nov, and cyr61 genes, which encode secreted proteins that contain conserved sequences and IGFBP motifs in their N-termini and bind IGFs with low affinity, represent more members of the IGFBP superfamily, along with the low-affinity mac25/IGFBP-7 (Yamanaka et al., J. Biol. Chem., 272: 30729-30734 (1997)) and the high-affinity IGFBPs 1-6. CTGF under this proposal would be designated IGFBP-8. Kim et al., Proc. Natl. Acad. Sci. USA, 94: 12981-12986 (1997).
Recently, a protein was found in the mouse designated ELM1 that is expressed in low metastatic cells. Hashimoto et al., J. Exp. Med., 187: 289-296 (1998). The elm1 gene, a mouse homologue of WISP-1 disclosed below, is another member of the CTGF, Cyr61/Cef10, and neuroblastoma overexpressed-gene family and suppresses in vivo tumor growth and metastasis of K-1735 murine melanoma cells. Another recent article on rCop-1, the rat orthologue of WISP-2 described below describes the loss of expression of this gene after cell transformation. Zhang et al., Mol. Cell. Biol., 18:6131-6141 (1998).
CTGF family members (with the exception of nov) are immediate early growth-responsive genes that are thought to regulate cell proliferation, differentiation, embryogenesis, and wound healing. Sequence homology among members of the CTGF gene family is high; however, functions of these proteins in vitro range from growth stimulatory (i.e., human CTGF) to growth inhibitory (i.e., chicken Nov and also possibly hCTGF). Further, some molecules homologous to CTGF are indicated to be useful in the prevention of desmoplasia, the formation of highly cellular, excessive connective tissue stroma associated with some cancers, and fibrotic lesions associated with various skin disorders such as scleroderma, keloid, eosinophilic fasciitis, nodular fasciitis, and Dupuytren""s contracture. Moreover, CTGF expression has recently been demonstrated in the fibrous stoma of mammary tumors, suggesting cancer stroma formation involves the induction of similar fibroproliferative growth factors as wound repair. Human CTGF is also expressed at very high levels in advanced atherosclerotic lesions, but not in normal arteries, suggesting it may play a role in atherosclerosis. Oemar and Luescher, supra. Therefore, molecules homologous to CTGF are of importance.
Extracellular and membrane-bound proteins play important roles in the formation, differentiation, and maintenance of multicellular organisms. The fate of many individual cells, e.g., proliferation, migration, differentiation, or interaction with other cells, is typically governed by information received from other cells and/or the immediate environment. This information is often transmitted by secreted polypeptides (for instance, mitogenic factors, survival factors, cytotoxic factors, differentiation factors, neuropeptides, and hormones), which are, in turn, received and interpreted by diverse cell receptors or membrane-bound proteins. These secreted polypeptides or signaling molecules normally pass through the cellular secretory pathway to reach their site of action in the extracellular environment, usually at a membrane-bound receptor protein.
Secreted proteins have various industrial applications, including use as pharmaceuticals, diagnostics, biosensors, and bioreactors. In fact, most protein drugs available at present, such as thrombolytic agents, interferons, interleukins, erythropoietins, colony stimulating factors, and various other cytokines, are secreted proteins. Their receptors, which are membrane-bound proteins, also have potential as therapeutic or diagnostic agents. Receptor immunoadhesins, for instance, can be employed as therapeutic agents to block receptor-ligand interaction. Membrane-bound proteins can also be employed for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction. Such membrane-bound proteins and cell receptors include, but are not limited to, cytokine receptors, receptor kinases, receptor phosphatases, receptors involved in cell-cell interactions, and cellular adhesin molecules like selectins and integrins. Transduction of signals that regulate cell growth and differentiation is regulated in part by phosphorylation of various cellular proteins. Protein tyrosine kinases, enzymes that catalyze that process, can also act as growth factor receptors. Examples include fibroblast growth factor receptor and nerve growth factor receptor.
Efforts are being undertaken by both industry and academia to identify new, native secreted and membrane-bound receptor proteins, particularly those having homology to CTGF. Many efforts are focused on the screening of mammalian recombinant DNA libraries to identify the coding sequences for novel secreted and membrane-bound receptor proteins. Examples of screening methods and techniques are described in the literature. See, for example, Klein et al., Proc. Natl. Acad. Sci., 93:7108-7113 (1996); and U.S. Pat. No. 5,536,637.
Wnts are encoded by a large gene family whose members have been found in round worms, insects, cartilaginous fish, and vertebrates. Holland et al., Dev. Suppl., 125-133 (1994). Wnts are thought to function in a variety of developmental and physiological processes since many diverse species have multiple conserved Wnt genes. McMahon, Trends Genet., 8: 236-242 (1992); Nusse and Varmus, Cell, 69: 1073-1087 (1992). Wnt genes encode secreted glycoproteins that are thought to function as paracrine or autocrine signals active in several primitive cell types. McMahon, supra (1992); Nusse and Varmus, supra (1992). The Wnt growth factor family includes more than ten genes identified in the mouse (Wnt-1, -2, -3A, -3B, -4, -5A, -5B, -6, -7A, -7B, -8A, -8B, -10B, -11, -12, and -13) (see, e.g., Gavin et al., Genes Dev., 4: 2319-2332 (1990); Lee et al., Proc. Natl. Acad. Sci. USA, 92: 2268-2272 (1995); Christiansen et al., Mech. Dev., 51: 341-350 (1995)) and at least nine genes identified in the human (Wnt-1, -2, -3, -5A, -7A, -7B, -8B, -10B, and -11) by cDNA cloning. See, e.g., Vant Veer et al., Mol.Cell.Biol., 4: 2532-2534 (1984).
The Wnt-1 proto-oncogene (int-1) was originally identified from mammary tumors induced by mouse mammary tumor virus (MMTV) due to an insertion of viral IDNA sequence. Nusse and Varmus, Cell, 31: 99-109 (1982). In adult mice, the expression level of Wnt-1 mRNA is detected only in the testis during later stages of sperm development. Wnt-1 protein is about 42 KDa and contains an amino-terminal hydrophobic region, which may function as a signal sequence for secretion (Nusse and Varmus, supra, 1992). The expression of Wnt-2/irp is detected in mouse fetal and adult tissues and its distribution does not overlap with the expression pattern for Wnt-1. Wnt-3 is associated with mouse mammary tumorigenesis. The expression of Wnt-3 in mouse embryos is detected in the neural tubes and in the limb buds. Wnt-5a transcripts are detected in the developing fore- and hind limbs at 9.5 through 14.5 days and highest levels are concentrated in apical ectoderm at the distal tip of limbs. Nusse and Varmus, supra (1992). Recently, a Wnt growth factor, termed Wnt-x, was described (WO95/17416) along with the detection of Wnt-x expression in bone tissues and in bone-derived cells. Also described was the role of Wnt-x in the maintenance of mature osteoblasts and the use of the Wnt-x growth factor as a therapeutic agent or in the development of other therapeutic agents to treat bone-related diseases.
Wnts may play a role in local cell signaling. Biochemical studies have shown that much of the secreted Wnt protein can be found associated with the cell surface or extracellular matrix rather than freely diffusible in the medium. Papkoff and Schryver, Mol. Cell. Biol., 10: 2723-2730 (1990); Bradley and Brown, EMBO J., 9: 1569-1575 (1990).
Studies of mutations in Wnt genes have indicated a role for Wnts in growth control and tissue patterning. In Drosophila, wingless (wg) encodes a Wnt-related gene (Rijsewik et al., Cell, 50: 649-657 (1987)) and wg mutations alter the pattern of embryonic ectoderm, neurogenesis, and imaginal disc outgrowth. Morata and Lawerence, Dev. Biol., 56: 227-240 (1977); Baker, Dev. Biol., 125: 96-108 (1988); Klingensmith and Nusse, Dev. Biol., 166: 396-414 (1994). In Caenorhabditis elegans, lin-44 encodes a Wnt homolog which is required for asymmetric cell divisions. Herman and Horvitz, Development, 120: 1035-1047 (1994). Knock-out mutations in mice have shown Wnts to be essential for brain development (McMahon and Bradley, Cell, 62: 1073-1085 (1990); Thomas and Cappechi, Nature, 346: 847-850 (1990)), and the outgrowth of embryonic primordia for kidney (Stark et al., Nature, 372: 679-683 (1994)), tail bud (Takada et al., Genes Dev., 8: 174-189 (1994)), and limb bud. Parr and McMahon, Nature, 374: 350-353 (1995). Overexpression of Wnts in the mammary gland can result in mammary hyperplasia (McMahon, supra (1992); Nusse and Varmus, supra (1992)), and precocious alveolar development. Bradbury et al., Dev. Biol., 170: 553-563 (1995).
Wnt-5a and Wnt-5b are expressed in the posterior and lateral mesoderm and the extraembryonic mesoderm of the day 7-8 murine embryo. Gavin et al., supra (1990). These embryonic domains contribute to the AGM region and yolk sac tissues from which multipotent hematopoietic precursors and HSCs are derived. Dzierzak and Medvinsky, Trends Genet., 11: 359-366 (1995); Zon et al., in Gluckman and Coulombel, ed., Colloque, INSERM, 235: 17-22 (1995), presented at the Joint International Workshop on Foetal and Neonatal Hematopoiesis and Mechanism of Bone Marrow Failure, Paris France, Apr. 3-6, 1995; Kanatsu and Nishikawa, Development, 122: 823-830 (1996). Wnt-5a, Wnt-10b, and other Wnts have been detected in limb buds, indicating possible roles in the development and patterning of the early bone microenvironment as shown for Wnt-7b. Gavin et al., supra (1990); Christiansen et al., Mech. Devel., 51: 341-350 (1995); Parr and McMahon, supra (1995).
The Wnt/Wg signal transduction pathway plays an important role in the biological development of the organism and has been implicated in several human cancers. This pathway also includes the tumor suppressor gene, APC. Mutations in the APC gene are associated with the development of sporadic and inherited forms of human colorectal cancer. The Wnt/Wg signal leads to the accumulation of beta-catenin/Armadillo in the cell, resulting in the formation of a bipartite transcription complex consisting of beta-catenin and a member of the lymphoid enhancer binding factor/T cell factor (LEF/TCF)HMG box transcription factor family. This complex translocates to the nucleus where it can activate expression of genes downstream of the Wnt/Wg signal, such as the engrailed and Ultrabithorax genes in Drosophila. The downstream target genes of Wnt-1 signaling in vertebrates that presumably function in tumorigenesis, however, are currently unknown.
For a most recent review on Wnt, see Cadigan and Nusse, Genes and Dev., 11: 3286-3305 (1997).
There is a need to elucidate the further members of the above families, including cell-surface molecules that may be tumor-specific antigens or proteins that serve a regulatory function in initiating the Wnt pathway of tumorigenesis. These would also include downstream components of the Wnt signaling pathway that are important to the transformed phenotype and the development of cancer.
Several putative Wnt-1-induced genes have been identified at the mRNA level in a high-throughput cDNA substraction experiment. Thus, applicants have identified novel cDNA clones (WISP1, WISP2, and WISP3) that encode novel polypeptides of the WISP family, designated as WISP-1, WISP-2, and WISP-3, respectively. This class of polypeptides was formerly referred to as Wnt-1-Induced Gene (WIG) polypeptides, with WISP-1 and WISP-2 formerly designated as WIG-1 and WIG-2, respectively. One of the cDNA clones encodes a novel polypeptide, human WISP-2, having homology to CTGF, wherein the polypeptide is designated in the present application as xe2x80x9chuman WISP-2xe2x80x9d or xe2x80x9cPRO261xe2x80x9d. The WISP-1 and WISP-3 molecules also have homology to CTGF.
In one embodiment, this invention provides isolated nucleic acid comprising DNA having at least about 600 nucleotides and at least about a 75% sequence identity to (a) a DNA molecule encoding a human WISP-1 polypeptide comprising the sequence of amino acids 23 to 367 of FIGS. 3A-3C (SEQ ID NO:3), or (b) the complement of the DNA molecule of (a). Preferably, this nucleic acid has at least one WISP biological activity. In a more preferred embodiment, this nucleic acid has at least about a 95% sequence identity to (a) a DNA molecule encoding a human WISP-1 polypeptide comprising the sequence of amino acids 23 to 367 of FIGS. 3A-3C (SEQ ID NO: 3), or (b) the complement of the DNA molecule of (a).
More preferred is the nucleic acid comprising DNA encoding a human WISP-1 polypeptide having amino acid residues 23 to 367 of FIGS. 3A-3C (SEQ ID NO:3), or DNA encoding a human WISP-1 polypeptide having amino acid residues 1 to 367 of FIGS. 3A-3C (SEQ ID NO:4), or the complement of either of the encoding DNAs. Further preferred is this nucleic acid comprising DNA encoding a human WISP-1 polypeptide having amino acid residues 23 to 367 or 1 to 367 of FIGS. 3A-3C except for an isoleucine residue at position 184 rather than a valine residue or a serine residue at position 202 rather than an alanine residue (SEQ ID NOS:5-8, respectively). Further preferred also is this nucleic acid comprising DNA encoding a human WISP-1 polypeptide having amino acid residues 23 to 367 or 1 to 367 of FIGS. 3A-3C except for an isoleucine residue at position 184 rather than a valine residue and a serine residue at position 202 rather than an alanine residue (SEQ ID NOS:21-22, respectively).
Also preferred is this nucleic acid comprising DNA encoding a mouse WISP-1 polypeptide having amino acid residues 23 to 367 of FIG. 1A-1B (SEQ ID NO:11), or DNA encoding a mouse WISP-1 polypeptide having amino acid residues 1 to 367 of FIGS. 1A-1B (SEQ ID NO:12), or the complement of either of the encoding DNAs.
Also provided by this invention is isolated nucleic acid comprising DNA having at least about 600 nucleotides and at least about a 85% sequence identity to (a) a DNA molecule encoding a mouse WISP-1 polypeptide comprising the sequence of amino acids 23 to 367 of FIGS. 1A-1B (SEQ ID NO:l1), or (b) the complement of the DNA molecule of (a). Preferably, this nucleic acid has at least one WISP biological activity. More preferably, this nucleic acid comprises DNA having at least about a 95% sequence identity to (a) a DNA molecule encoding a mouse WISP-1 polypeptide comprising the sequence of amino acids 23 to 367 of FIGS. 1A-1B (SEQ ID NO:11), or (b) the complement of the DNA molecule of (a). Preferably, this nucleic acid has at least one WISP biological activity. More preferably, this nucleic acid comprises DNA having at least about a 95% sequence identity to (a) a DNA molecule encoding a mouse WISP-1 polypeptide comprising the sequence of amino acids 23 to 367 of FIGS. 1A-1B (SEQ ID NO:11), or (b) the complement of the DNA molecule of (a).
In another preferred embodiment, the invention provides an isolated nucleic acid comprising DNA having at least about 600 nucleotides and at least about a 75% sequence identity to (a) a DNA molecule encoding the same full-length polypeptide encoded by the human WISP-1 polypeptide cDNA in ATCC Deposit No. 209533_(pRK5E.h.WISP-1.568.38), or (b) the complement of the DNA molecule of (a). This nucleic acid preferably comprises DNA having at least about 600 nucleotides and at least about a 95% sequence identity to (a) a DNA molecule encoding the same full-length polypeptide encoded by the human WISP-1 polypeptide cDNA in ATCC Deposit No. 209533 (pRK5E.h.WISP-1.568.38), or (b) the complement of the DNA molecule of (a).
In another aspect, the invention provides a process for producing a WISP-1 polypeptide comprising culturing a host cell comprising the above nucleic acid under conditions suitable for expression of the WISP-1 polypeptide and recovering the WISP-1 polypeptide from the cell culture. Additionally provided is an isolated WISP-1 polypeptide encoded by the above nucleic acid, including where the polypeptide is human WISP-1 or mouse WISP-1.
In another embodiment, the invention provides isolated nucleic acid comprising SEQ ID NO:23, 24, 25, 26, 27, 28, or 29, and an isolated WISP-1 polypeptide encoded by such a nucleic acid.
Also provided by this invention is an isolated nucleic acid having at least about 600 nucleotides and produced by hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a human WISP-1 polypeptide comprising the sequence of amino acids 23 to 367 of FIGS. 3A-3C (SEQ ID NO:3), or (b) the complement of the DNA molecule of (a), and, if the test DNA molecule has at least about a 75% sequence identity to (a) or (b), isolating the test DNA molecule.
Further provided is a polypeptide produced by (i) hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a human WISP-1 polypeptide comprising the sequence of amino acids 23 to 367 of FIGS. 3A-3C (SEQ ID NO:3), or (b) the complement of the DNA molecule of (a), and if the test DNA molecule has at least about a 75% sequence identity to (a) or (b), (ii) culturing a host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, and (iii) recovering the polypeptide from the cell culture.
In another aspect, the invention provides isolated nucleic acid comprising DNA having at least about an 80% sequence identity to (a) a DNA molecule encoding a human WISP-2 polypeptide comprising the sequence of amino acids 24 to 250 of FIGS. 4A-4B (SEQ ID NO:15), or (b) the complement of the DNA molecule of (a). Preferably, this nucleic acid has at least one WISP biological activity. Also, preferably this nucleic acid comprises DNA having at least about a 95% sequence identity to (a) a DNA molecule encoding a human WISP-2 polypeptide comprising the sequence of amino acids 24 to 250 of FIGS. 4A-4B (SEQ ID NO:15), or (b) the complement of the DNA molecule of (a). In another preferred embodiment, this nucleic acid comprises DNA encoding a human WISP-2 polypeptide having amino acid residues 24 to 250 of FIGS. 4A-4B (SEQ ID NO:15), or DNA encoding a human WISP-2 polypeptide having amino acid residues 1 to 250 of FIGS. 4A-4B (SEQ ID NO:16), or a complement of either of the encoding DNAs.
In another aspect, the invention provides isolated nucleic acid comprising DNA having at least about an 80% sequence identity to (a) a DNA molecule encoding a human WISP-2 polypeptide comprising the sequence of amino acids 1 to 250 of FIGS. 4A-4B (SEQ ID NO:16), or (b) the complement of the DNA molecule of (a).
In another aspect, the invention provides isolated nucleic acid comprising DNA having at least about 500 nucleotides and at least about an 80% sequence identity to (a) a DNA molecule encoding a mouse WISP-2 polypeptide comprising the sequence of amino acids 24 to 251 of FIGS. 2A-2B (SEQ ID NO:19), or (b) the complement of the DNA molecule of (a). In a preferred embodiment, this nucleic acid comprises DNA having at least about a 95% sequence identity to (a) a DNA molecule encoding a mouse WISP-2 polypeptide comprising the sequence of amino acids 24 to 251 of FIGS. 2A-2B (SEQ ID NO:19), or (b) the complement of the DNA molecule of (a). More preferably, the nucleic acid comprises DNA encoding a mouse WISP-2 polypeptide having amino acid residues 24 to 251 of FIGS. 2A-2B (SEQ ID NO:19), or DNA encoding a mouse WISP-2 polypeptide having amino acid residues 1 to 251 of FIGS. 2A-2B (SEQ ID NO:20), or the complement of either of these encoding DNAs.
In a further aspect, the invention provides isolated nucleic acid comprising DNA having at least about 500 nucleotides and at least about an 80% sequence identity to (a) a DNA molecule encoding a mouse WISP-2 polypeptide comprising the sequence of amino acids 1 to 251 of FIGS. 2A-2B (SEQ ID NO:20), or (b) the complement of the DNA molecule of (a).
In yet another aspect, the invention provides an isolated nucleic acid comprising DNA having at least about 400 nucleotides and at least about a 75% sequence identity to (a) a DNA molecule encoding the same full-length polypeptide encoded by the human WISP-2 polypeptide cDNA in ATCC Deposit No. 209391 (DNA33473), or (b) the complement of the DNA molecule of (a). Preferably, this nucleic acid comprises DNA having at least about a 95% sequence identity to (a) a DNA molecule encoding the same full-length polypeptide encoded by the human WISP-2 polypeptide cDNA in ATCC Deposit No. 209391 (DNA33473), or (b) the complement of the DNA molecule of (a).
In another embodiment, this invention provides an isolated nucleic acid comprising the nucleotide sequence of the full-length coding sequence of clone UNQ228 (DNA33473) deposited under accession number ATCC 209391.
In another aspect, the invention provides a process for producing a WISP-2 polypeptide comprising culturing a host cell comprising the above nucleic acid under conditions suitable for expression of the WISP-2 polypeptide and recovering the WISP-2 polypeptide from the cell culture. Additionally provided is a WISP-2 polypeptide encoded by the isolated nucleic acid, including where the polypeptide is human WISP-2 or mouse WISP-2. In a specific embodiment of this, the invention provides isolated native-sequence human WISP-2 polypeptide comprising amino acid residues 1 to 250 of FIGS. 4A-4B (SEQ ID NO:16) or comprising amino acid residues 24 to 250 of FIGS. 4A-4B (SEQ ID NO:15).
In a further embodiment, the invention provides an isolated nucleic acid having at least about 400 nucleotides and produced by hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a human WISP-2 polypeptide comprising the sequence of amino acids 24 to 250 of FIGS. 4A-4B (SEQ ID NO:15), or (b) the complement of the DNA molecule of (a), and, if the test DNA molecule has at least about a 75% sequence identity to (a) or (b), isolating the test DNA molecule.
In a still further embodiment, the invention provides a polypeptide produced by (i) hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a human WISP-2 polypeptide comprising the sequence of amino acids 24 to 250 of FIGS. 4A-4B (SEQ ID NO:15), or (b) the complement of the DNA molecule of (a), and if the test DNA molecule has at least about a 75% sequence identity to (a) or (b), (ii) culturing a host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, and (iii) recovering the polypeptide from the cell culture.
In yet another embodiment, the invention provides isolated nucleic acid comprising DNA having a 100% sequence identity in more than about 500 nucleotides to (a) a DNA molecule encoding a human WISP-3 polypeptide comprising the sequence of amino acids 34 to 372 of FIGS. 6A-6C (SEQ ID NO:32), or (b) the complement of the DNA molecule of (a). Preferably, this nucleic acid has at least one WISP biological activity. Preferably, this nucleic acid comprises DNA encoding a human WISP-3 polypeptide having amino acid residues 34 to 372 of FIGS. 6A-6C (SEQ ID NO:32) or amino acids 1 to 372 of FIGS. 6A-6C (SEQ ID NO:33), or the complement thereof.
In a still further embodiment, the invention provides an isolated nucleic acid comprising DNA having a 100% sequence identity in more than about 500 nucleotides to (a) a DNA molecule encoding the same full-length polypeptide encoded by the human WISP-3 polypeptide cDNA in ATCC Deposit No. 209706 (DNA56350-1176-2), or (b) the complement of the DNA molecule of (a). A still further aspect of the invention involves a process for producing a WISP-3 polypeptide comprising culturing a host cell comprising WISP-3-encoding nucleic acid under conditions suitable for expression of the WISP-3 polypeptide and recovering the WISP-3 polypeptide from the cell culture.
Further provided is an isolated WISP-3 polypeptide encoded by the WISP-3-encoding nucleic acid. Preferably, this polypeptide is human WISP-3.
In another embodiment, the invention provides an isolated nucleic acid produced by hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a human WISP-3 polypeptide comprising the sequence of amino acids 34 to 372 of FIGS. 6A-6C (SEQ ID NO:32), or (b) the complement of the DNA molecule of (a), and, if the test DNA molecule has a 100% sequence identity to (a) or (b) in more than about 500 nucleotides, isolating the test DNA molecule.
Also provided is a polypeptide produced by (i) hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a human WISP-3 polypeptide comprising the sequence of amino acids 34 to 372 of FIGS. 6A-6C (SEQ ID NO:32), or (b) the complement of the DNA molecule of (a), and if the test DNA molecule has a 100% sequence identity to (a) or (b) in more than about 500 nucleotides, (ii) culturing a host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, and (iii) recovering the polypeptide from the cell culture.
In yet another embodiment, the invention provides isolated nucleic acid comprising DNA having a 100% sequence identity in more than about 400 nucleotides to (a) a DNA molecule encoding a human WISP-3 polypeptide comprising the sequence of amino acids 16 to 355 of FIGS. 7A-7C (SEQ ID NO:36), or (b) the complement of the DNA molecule of (a). Preferably, this nucleic acid has at least one WISP biological activity. Preferably, this nucleic acid comprises DNA encoding a human WISP-3 polypeptide having amino acid residues 16 to 355 of FIGS. 7A-7C (SEQ ID NO:36), or amino acid residues 1 to 355 of FIGS. 7A-7C (SEQ ID NO:37) or the complement thereof.
In a still further embodiment, the invention provides an isolated nucleic acid comprising DNA having a 100% sequence identity in more than about 400 nucleotides to (a) a DNA molecule encoding the same full-length polypeptide encoded by the human WISP-3 polypeptide cDNA in ATCC Deposit No. 209707 (DNA58800-1176-2), or (b) the complement of the DNA molecule of (a).
A still further aspect of the invention involves a process for producing a WISP-3 polypeptide of FIGS. 7A-7C comprising culturing a host cell comprising WISP-3-encoding nucleic acid under conditions suitable for expression of the WISP-3 polypeptide and recovering the WISP-3 polypeptide from the cell culture.
Further provided is an isolated WISP-3 polypeptide of FIGS. 7A-7C encoded by the WISP-3-encoding nucleic acid. Preferably, this polypeptide is human WISP-3.
In another embodiment, the invention provides an isolated nucleic acid produced by hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a human WISP-3 polypeptide comprising the sequence of amino acids 16 to 355 of FIGS. 7A-7C (SEQ ID NO:36), or (b) the complement of the DNA molecule of (a), and, if the test DNA molecule has a 100% sequence identity to (a) or (b) in more than about 400 nucleotides, isolating the test DNA molecule.
Also provided is a polypeptide produced by (i) hybridizing a test DNA molecule under stringent conditions with (a) a DNA molecule encoding a human WISP-3 polypeptide comprising the sequence of amino acids 16 to 355 of FIGS. 7A-7C (SEQ ID NO:36), or (b) the complement of the DNA molecule of (a), and if the test DNA molecule has a 100% sequence identity to (a) or (b) in more than about 400 nucleotides, (ii) culturing a host cell comprising the test DNA molecule under conditions suitable for expression of the polypeptide, and (iii) recovering the polypeptide from the cell culture.
Preferably the complements of the DNA molecules herein remain stably bound to the primary sequence under at least moderate, and optionally, under high stringency conditions.
Also provided are vectors comprising the above nucleic acids, host cells comprising the vector, preferably wherein the cell is a Chinese hamster ovary (CHO) cell, an E. coli cell, a baculovirus-infected cell, or a yeast cell.
Additionally provided are a chimeric molecule comprising one of the above polypeptides or an inactivated variant thereof, fused to a heterologous amino acid sequence, wherein the heterologous amino acid sequence may be, for example, an epitope tag sequence, a polyamino acid such as poly-histidine, or an immunoglobulin constant region (Fc). Also provided is an antibody which specifically binds to one of the above polypeptides, wherein the antibody can be a monoclonal antibody.
Further provided are a composition comprising one of the above polypeptides and a carrier therefor, and a composition comprising an antagonist to one of the polypeptides and a carrier therefor. In one such embodiment, the invention provides a composition comprising a WISP-1, WISP-2, or WISP-3 polypeptide and a pharmaceutically acceptable carrier. Preferably, the polypeptide is a human polypeptide. Also, preferably, these compositions may also comprise a chemotherapeutic agent or growth-inhibitory agent.
In another aspect, the invention provides a pharmaceutical product comprising:
(a) the composition comprising WISP-1, WISP-2, or WISP-3 polypeptide and a pharmaceutically acceptable carrier;
(b) a container containing said composition; and
(c) a label affixed to said container, or a package insert included in said pharmaceutical product referring to the use of said WISP-1, WISP-2, or WISP-3 polypeptide in the treatment of a WISP-related disorder.
In yet another embodiment, the invention provides a method for treating a WISP-related disorder in a mammal comprising administering to the mammal an effective amount of any of the above compositions, including the composition of a WISP-1, WISP-2, or WISP-3 polypeptide in a pharmaceutically acceptable carrier, and including the composition of an antagonist to a WISP-1, WISP-2, or WISP-3 polypeptide in a pharmaceutically acceptable carrier. Preferably, the disorder is a malignant disorder or arteriosclerosis. More preferably, the malignant disorder is breast cancer, ovarian cancer, colon cancer, or melanoma. Also, preferably the mammal is human. In another preferred embodiment, the WISP-1, WISP-2, or WISP-3 polypeptide is administered in combination with a chemotherapeutic agent, a growth inhibitory agent, or a cytotoxic agent.
In another embodiment, the invention supplies a process for diagnosing a disease or a susceptibility to a disease related to a mutation in a nucleic acid sequence encoding a WISP-1, WISP-2, or WISP-3 polypeptide comprising:
(a) isolating a nucleic acid sequence encoding a WISP-1, WISP-2, or WISP-3 polypeptide from a sample derived from a host; and
(b) determining a mutation in the nucleic acid sequence encoding a WISP-1, WISP-2, or WISP-3 polypeptide.
In another embodiment, the invention provides a method of diagnosing a WISP-related disorder in a mammal comprising detecting the level of expression of a gene encoding a WISP-1, WISP-2, or WISP-3 polypeptide (a) in a test sample of tissue cells obtained from the mammal, and (b) in a control sample of known normal tissue cells of the same cell type, wherein a higher or lower expression level in the test sample indicates the presence of a WISP-related dysfunction in the mammal from which the test tissue cells were obtained. Preferably, such a disorder is a type of cancer and a higher expression level in the test sample indicates the presence of a tumor in the mammal.
In a still further embodiment, the invention provides an isolated antibody binding a WISP-1, WISP-2, or WISP-3 polypeptide. Preferably, the antibody induces death of a cell overexpressing a WISP-1, WISP-2, or WISP-3 polypeptide, more preferably a cancer cell. Also preferred is an antibody that binds to a human WISP-1, WISP-2, or WISP-3 polypeptide, and is a human or humanized antibody. More preferred is a monoclonal antibody, still more preferred, a monoclonal antibody that has complementary-determining regions and constant immunoglobulin regions, and in other embodiments is an antibody fragment, a single-chain antibody, or an anti-idiotypic antibody. In addition, the antibody is suitably labeled with a detectable label or immobilized on a solid support.
Also provided is a composition comprising an antibody to a WISP-1, WISP-2, or WISP-3 polypeptide in admixture with a pharmaceutically accetable carrier. Preferably, the antibody is to a human WISP-1, WISP-2, or WISP-3 polypeptide, and is a human or humanized antibody, most preferably a monoclonal antibody against human WISP-1. Further, the composition may comprise a growth-inhibitory amount of said antibody.
In another embodiment, the invention provides a method for treating cancer in a mammal comprising administering to the mammal an effective amount of the above antibody composition. In a preferred aspect of this method, the cancer is colon cancer, the antibody is against human WISP-1 and is a humanized or human monoclonal antibody, and the mammal is human.
In another aspect, the invention provides a method for treating a WISP-related disorder in a mammal comprising administering to the mammal an effective amount of a composition comprising an antagonist to a WISP-1, WISP-2, or WISP-3 polypeptide in a pharmaceutically acceptable carrier.
In a further aspect, the invention provides a method for inhibiting the growth of tumor cells comprising exposing a cell that overexpresses a Wnt-1-induced gene to an effective amount of an antagonist that inhibits the expression or activity of a WISP-1, WISP-2, or WISP-3 polypeptide.
A further aspect entails a method for inhibiting the growth of tumor cells comprising exposing said cells to an effective amount of the composition with the growth-inhibiting amount of an anti-WISP-1, anti-WISP-2, or anti-WISP-3 antibody in admixture with the carrier. In a preferred aspect of this method, the tumor cells are colon cancer cells, the antibody is against human WISP-1 and is a humanized or human monoclonal antibody, and the mammal is human.
Also provided herein is a kit comprising one of the above WISP polypeptides or WISP antagonists, such as anti-WISP antibodies, and instructions for using the polypeptide or antagonist to detect or treat a WISP-related disorder, such as cancer induced by Wnt. One such preferred kit is a cancer diagnostic kit comprising an anti-WISP-1, anti-WISP-2, or anti-WISP-3 antibody and a carrier in suitable packaging. Preferably, this kit further comprises instructions for using said antibody to detect the WISP-1, WISP-2, or WISP-3 polypeptide.
Also provided is a method for inducing cell death comprising exposing a cell which is induced by Wnt to an effective amount of one of the above WISP polypeptides or WISP antagonists, such as anti-WISP antibodies. Preferably, such cell is a cancer cell. More preferably, the cell is in a mammal, more preferably a human. In addition, an effective amount of another chemotherapeutic antibody is used in the exposure of the cell, such as an anti-ErbB2 antibody. Further, optionally the method comprises exposing the cell to a chemotherapeutic agent, a growth-inhibitory agent, or radiation. Optionally, the cell is exposed to the growth-inhibitory agent prior to exposure to the antibody.
In a further aspect, the invention provides an article of manufacture, comprising:
a container;
a label on the container; and
a composition comprising an active agent contained within the container;
wherein the composition is effective for inducing cell death or inhibiting the growth of tumor cells, the label on the container indicates that the composition can be used for treating conditions characterized by overinduction of Wnt or a WISP-related disorder or by overexpression of a WISP-1, WISP-2, or WISP-3 polypeptide, and the active agent in the composition is an antagonist to one of the polypeptides, that is, an agent that inhibits the expression and/or activity of the WISP-1, WISP-2, or WISP-3 polypeptide. Preferably, the active agent in such article of manufacture is an anti-WISP-1, anti-WISP-2, or anti-WISP-3 antibody, and the label on the container indicates that the composition can be used for treating a WISP-related disorder.
In another embodiment, the invention provides a process for identifying agonists to a WISP-1, WISP-2, or WISP-3 polypeptide comprising:
(a) contacting cells and a compound to be screened under conditions suitable for the stimulation of cell proliferation by the polypeptide; and
(b) measuring the proliferation of the cells to determine if the compound is an effective agonist.
Additionally, the invention provides an agonist to a WISP-1, WISP-2, or WISP-3 polypeptide identified by the above process.
Further, the invention provides a method for identifying a compound that inhibits the expression or activity of a WISP-1, WISP-2, or WISP-3 polypeptide, comprising contacting a candidate compound with a WISP-1, WISP-2, or WISP-3 polypeptide under conditions and for a time sufficient to allow the compound and polypeptide to interact. In a preferred embodiment, this method comprises the steps of:
(a) contacting cells and a compound to be screened in the presence of the WISP-1, WISP-2, or WISP-3 polypeptide under conditions suitable for the stimulation of cell proliferation by polypeptide; and
(b) measuring the proliferation of the cells to determine if the compound is an effective antagonist.
Further, a compound identified by this method is provided.
In another aspect, this invention provides a compound that inhibits the expression or activity of a WISP-1, WISP-2, or WISP-3 polypeptide.
In another embodiment, the invention provides a method for determining the presence of a WISP-1, WISP-2, or WISP-3 polypeptide comprising exposing a cell suspected of containing the WISP-1, WISP-2, or WISP-3 polypeptide to an anti-WISP-1, anti-WISP-2, or anti-WISP-3 antibody and determining binding of said antibody to said cell.
In another preferred embodiment, the invention provides a method of diagnosing a WISP-related disorder in a mammal comprising (a) contacting an anti-WISP-1, anti-WISP-2, or anti-WISP-3 antibody with a test sample of tissue cells obtained from the mammal, and (b) detecting the formation of a complex between the anti-WISP-1, anti-WISP-2, or anti-WISP-3 antibody and the WISP-1, WISP-2, or WISP-3 polypeptide in the test sample. Preferably, said test sample is obtained from an individual suspected to have neoplastic cell growth or proliferation. Also, preferably the antibody is labeled with a detectable label and/or is immobilized on a solid support.